MCDB 428 Exam I Key Winter 2008

MCDB 428 Exam I Key Winter 2008 - Name Your GSI MCDB 428...

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Name ____________________________________ Your GSI_________________ MCDB 428 Cell Biology January 28, 2008 Exam I The exam has 12 questions from lectures 1-9 (the whole exam is 100 pts) Remember to pace yourself; the exam time is 2 hours For the short answer questions, you can continue on the back of the page if necessary Please write your name on the top right hand corner of each page The exam proctors will not answer questions during the test unless they concern typos, or unless essential information is missing Points will be deducted if what you write is incorrect whether or not it is relevant Good Luck! 1
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Name ____________________________________ 1. (12 pts) The purification of a microtubule motor protein has been undertaken. A partially purified cell fraction containing the motor protein was subjected to an additional purification step using rate-zonal centrifugation. Fractions were collected from the top of the sucrose density gradient (fraction 1) to the bottom of the gradient (fraction 41). Aliquots of each fraction collected were subjected to SDS gel electrophoresis. Fractions 21 to 41 from the gradient are shown on the gel below, which was stained with Coomassie blue. a). What differing properties of proteins likely account for the bands on the gel observed in lanes 21–26 versus those observed in lanes 27–36? The proteins in fractions 21-26 must have a smaller molecular radius (Mr) than those in fractions 27-36. (4 pts) (a) Based on SDS-PAGE, the proteins in fractions 21-26 have higher molecular weight than the proteins in fractions 27-36. (1point ) (b) Rate zonal centrifugation differentiates protein based on their Mr/MW/density and size. (3 points) b). In the initial experiment the sample was spun for 30 minutes. If you performed the experiment again, but this time the sample was spun for 2 hours would you expect the upper and lower proteins bands to be present in the same fractions or different ones? Why? The proteins should move to lower fractions in the sucrose gradient because they are denser than the sucrose and therefore have not reached equilibrium. (4 pts) (a) If the answer said that additional spinning resulted in increased resolution then this was worth (2points) (b) If the answer said that the proteins were in different fractions but said that the proteins had reached equilibrium/same fractions (0 point) 2
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Name ____________________________________ c). Molecular motors are ATPases that use energy of hydrolysis of ATP to generate a motive (movement) force and this is accompanied with a change in protein conformation. You treat your partially purified motor protein sample with the non-hydrolyzable ATP analog, ATP γ S, and again perform rate zonal centrifugation. After performing SDS gel electrophoresis you now observe that the lower protein band is present in Fractions 23-29. From other experiments you have determined that this protein does not change is oligomeric state or interact with other proteins in the presence of ATP γ S. Why does this protein now sediment more slowly than before through the sucrose density gradient?
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